In the early 1980's, melanotransferrin (MTf) was identified as an oncofetal antigen that was either not expressed, or only slightly expressed in normal tissues, but was found in much larger amounts in neoplastic cells (especially malignant melanoma cells) and fetal tissues (Woodbury, et al., P.N.A.S. USA, 77:2183-2187 (1980)). More recently, there have been additional reports of human MTf being identified in normal tissues, including sweat gland ducts, liver endothelial cells and the endothelium and reactive microglia of the brain (Jefferies, et al., Brain Res., 712:122-126 (1996); and Rothenberger, et al., Brain Res., 712:117-121 (1996)). Interestingly, normal serum contains very low levels of soluble circulating MTf, but increased soluble serum MTf has been found in patients with advanced Alzheimer's Disease (Kennard, et al., Nat. Med., 2:1230-1235 (1996); U.S. Pat. No. 5,981,194)
The biochemical role and metabolism of MTf has proven difficult to elucidate. Based on appearances, MTf is deceptively similar to transferrin (Tf) and lactotransfernin (lactoferrin or Lf). In humans, these proteins share a 37-39% amino acid sequence homology. In particular, each of these proteins reversibly binds iron, and their N-terminal iron binding domains are quite similar (Baker, et al., TIBS, 12:350-353 (1987)).
However, functional parallels between these proteins have not been confirmed. For one thing, unlike Tf and Lf, MTf exists in both a membrane bound form and a serum soluble form. Further, in contrast to Tf and Lf, no cellular receptor for MTf has been identified. Serum soluble Tf is known to be taken into cells in an energy-dependent process mediated by the transferrin receptor (Tf-R) (Cook, et al., Annu. Rev. Med., 44:63-74 (1993)). Lf internalization is also likely to be mediated by a receptor mediated process (Fillebeen, et al., J. Biol. Chem., 274(11):7011-7017 (1999)). Two known receptors for Lf are LRP1 and RAGE, although others may exist (Meilinger, et al., FEBS Letters, 360:70-74 (1995); Schmidt, J. Biol. Chem., 269(13):9882-9888 (1994)).
Although it has been postulated that MTf is an alternate ligand for Tf-R (see, U.S. Pat. No. 5,981,194), no published data confirms this finding. Further, although various studies have confirmed iron transport into cells by membrane bound MTf, it occurs only in cells where membrane bound MTf is overexpressed well beyond physiological levels (Richardson, Eur. J. Biochem., 267:1290-1298 (2000)).
Therapeutic and diagnostic agents conjugated to soluble MTf are the basis of recently filed U.S. Provisional Patent Application Nos. 60/226,242 and 60/226,254, the teachings of which are incorporated herein by reference for all purposes.
The blood-brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of pharmacological agents to cerebral tissues, necessitating the use of vectors for their transit. Blood-brain barrier (BBB) permeability is frequently a rate-limiting factor for the penetration of drugs or peptides into the central nervous system (CNS) (see Pardridge, W. M. J. Neurovirol. 5: 556-569 (1999); Bickel, U., Yoshikawa, T. & Pardridge, W. M. Adv. Drug Deliv. Rev. 46: 247-279 (2001). The brain is shielded against potentially toxic substances by the BBB, which is formed by brain capillary endothelial cells that are closely sealed by tight junctions. In addition, brain capillaries possess few fenestrae and few endocytic vesicles, compared to the capillaries of other organs (see Pardridge, W. M. J. Neurovirol. 5: 556-569 (1999)). There is little transit across the BBB of large, hydrophilic molecules aside from some specific proteins such as transferrin, lactoferrin and low-density lipoproteins, which are taken up by receptor-mediated endocytosis (see Pardridge, W. M. J. Neurovirol. 5: 556-569 (1999); Tsuji, A. & Tamai, I. Adv. Drug Deliv. Rev. 36: 277-290 (1999); Kusuhara, H. & Sugiyama, Y. Drug Discov. Today 6:150-156 (2001); Dehouck, B. et al. J. Cell. Biol. 138: 877-889 (1997); and Fillebeen, C. et al. J. Biol. Chem. 274: 7011-7017 (1999).
In order to understand and improve the delivery of therapeutic agents into cells, it is highly desirable to understand the receptors and metabolic basis of MTf activity. It is an object of this invention to identify the receptor for MTf, and to provide methods and compounds for improving the delivery of therapeutic and diagnostic agents into cells, in particular, therapeutic and diagnostic agents conjugated to MTf, and particularly their delivery across the blood-brain barrier.